Oral tolerance has been associated with the control of experimental autoimmune diseases including experimental autoimmune encephalomyelitis, the animal model of human multiple sclerosis (MS). In tolerance, low antigen dose appears to suppress, whereas higher doses induce clonal anergy. Tolerance depends on polarization to a Th2 (IL-4, IL-10) and/or Th3 (TGF-β) phenotype. Regulatory CD4+CD25+ Treg cells, as well as IL-4, IL-10, anti-IL-12p40, TGF-β, and anti-CD40 ligand enhance tolerance (Weber, et al. (2007) Nat. Med. 13:935). Oral antigens such as MBP or glatiramer acetate, an FDA approved therapy for relapsing-remitting multiple sclerosis have been shown to suppresses EAE (Weber, et al. (2007) supra).
Studies have demonstrated that tolerance induction through oral immunization with foreign antigens can control the development of autoimmune diseases. Oral immunization with a single dose of an attenuated strain of Salmonella Typhimurium expressing the CFA/I fimbriae of Enterotoxigenic E. coli confers prophylactic and therapeutic protection against EAE in SJL mice (Jun, et al. (2005) J. Immunol. 175:6733). Salmonella-CFA/I elicits FoxP3+CD4+CD25+ Treg cells that produce TGF-β (Ochoa-Reparaz (2007) J. Immuno. 178:1791), and although a switch to Th-2 type immune responses played a role in diminishing EAE, the role of Treg cells was clearly predominant. Adoptive transfer of Salmonella-CFA/I-induced Treg cells, but not naïve Treg cells, protected against EAE, suggesting that the immunization with Salmonella, an irrelevant non-self bacterial antigen, induced and expanded Treg cells in mice that were protective against EAE (Ochoa-Reparaz (2007) supra).
Similarly, it has been shown that dextran sodium sulphate-induced colitis can be suppressed by oral administration of antigens from normal intestinal flora anaerobes (Verdù, et al. (2000) Clin. Exp. Immunol. 120:46-50).
Although mammals are born sterile, microorganisms soon colonize their mucosal surfaces after birth. The colonization of the mucosal surface of the gut evolves into a highly diverse endogenous microflora population composed of over 1013 resident bacteria, creating a relationship that confers benefits to both microorganisms and host (Hooper & Gordon (2001) Science 292:1115). However, this environment can be shared by multiple pathogens that utilize the mucosa as invasion and infection sites. It is the role of the immune system to concurrently control the responses to commensal and pathogenic organisms. It is possible that changes in the microbial composition of the intestinal compartments modify the phenotype, proliferation and functional capacity of regulatory T cells. In this regard, it has been suggested that the significant involvement of the gut microbiota in human health and disease indicates that manipulation of commensal microbial composition through combinations of antibiotics, probiotics and prebiotics could be a novel therapeutic approach (Jia, et al. (2008) Nat. Rev. Drug Discov. 7(2):123-9).
The role of regulatory T cells in the induction of tolerance has been analyzed (Weiner (2001) Microbes Infect. 3:947; Zhang & Zhao (2007) J. Cell Physiol. 211:590; Pascual, et al. (2007) Endocr. Metab. Immune Disord. Drug Targets 7:203; Sakaguchi, et al. (2007) Eur. J. Immunol. 37 Suppl 1:S11). Retinoic acid expressed by CD103+ gut-derived dendritic cells in the presence of TGF-β appears essential to the conversion of naïve CD4+CD25-effector T cells into a regulatory FoxP3+Treg cells subpopulation (Weiner (2001) supra; Zhang & Zhao (2007) supra; Pascual, et al. (2007) supra). Treg cells expressing the surface trafficking molecule α4β7 migrate specifically to the gut mucosa linking the expression of retinoic acid to the conversion and migration of Treg cells. It has been demonstrated that the metabolism of vitamin A into retinoic acid appears to depend on the presence of commensal bacteria (Schambach, et al. (2007) Eur. J. Immunol. 37:2396). Absence of bacteria in germ-free mice (axenic) that are born and raised in sterile isolators demonstrates that the presence of commensal bacteria is essential for normal immune development. Alterations in the immune profile in these mice exhibit a default Th2 bias and a significant reduction in proinflammatory IL-17-producing CD4+ T cells compared to mice with an intact communal gut bacterial profile (Niess, et al. (2008) J. Immunol. 180:559).
Bacteroides fragilis, a gram-negative anaerobe, is the prototypic member of the microflora in the normal mammalian gut (Niess, et al. (2008) supra). Genomic analysis has shown that B. fragilis is able to produce eight different capsular polysaccharides (PSA-PSH) with on-off phase variable phenotypes that may be involved in the broad adaptability of this bacteria to different mammalian hosts (Krinos, et al. (2001) Nature 414:555). Studies have demonstrated that at least one of these polysaccharides, the zwitterrionic polymer PSA is able to induce the activation and proliferation of CD4+ T cells. Moreover, PSA controls the Th1/Th2 physiologic balance in germ-free animals colonized with Bacteroides fragilis and immunized with purified PSA (Mora, et al. (2003) Nature 424:88; Mora, et al. (2006) Science 314:1157). Gut colonization in germ-free mice demonstrated that PSA is able to interact with TLR2 signaling in dendritic cells (DCs) and stimulate T cell activation (Mora, et al. (2003) supra). Immunization with purified PSA expanded T cell populations in both germ-free and intact mice, increased MHC class II expression among CD11c+ DCs and the expression of CD80 and CD86 (Mora, et al. (2006) supra). Interestingly, germ-free animals have a default Th2 bias with increased production of IL-4 and low levels of IFN-γ as opposed to intact animals (Massacesi, et al. (1987) J. Neurol. Sci. 80:55; Macpherson, et al. (2001) Microbes Infect. 3:1021; Iwata, et al. (2004) Immunity 21:527). When germ-free mice were infected with B. fragilis, the IL-4 levels were significantly reduced while IFN-γ production was restored (Mora, et al. (2006) supra). Infection of germ-free mice with B. fragilis deficient in PSA did not provoke this cytokine profile switching (Mora, et al. (2006) supra).
It has been observed that retinoic acid induces the homing of leukocytes to the gut. The peripheral homing preferences of T cells to migrate to a specific tissue are imprinted by DCs from that tissue during antigen presentation (Mora, et al. (2003) supra). DCs from the gut Mesenteric lymph nodes (MLN) and Peyer's patches (PP) induces the gut-homing markers α4β7 and CCR9 on T cells, while DCs from peripheral lymph nodes (PLN) induced, preferentially, the skin homing receptors P- and E-selectin ligands. Based on this observation and the observation that Vitamin A deficiency impairs gut immunity, the function of retinoic acid on the expression of homing receptors on T cells has been evaluated (Mora, et al. (2003) supra). In these experiments, CD4+ T cells were stimulated with αCD3 and αCD28 in the presence or absence of retinoic acid. The addition of retinoic acid enhanced the expression of α4β7 and the mRNA levels of CCR9, together with the suppression of E-selectin ligand expression. This effect was not impaired when T cells were cultured under Th1 or Th2 skewing conditions. In addition, CD4+ T cells cultured in the presence of retinoic acid showed increased migration to the CCR9 ligand, TECK, when tested in trans-well experiments. Performing competitive homing experiments in vivo, it was observed that retinoic acid-treated CD4+ T cells migrated preferentially to the gut, in comparison with untreated CD4+ T cells. Furthermore, the potential population of cells synthesizing retinoic acid in vivo was determined by the expression of the enzyme RALDH. It was observed that MLN- and PP-DCs, but not PLN-DCs, express RALDH. In addition, MLN- and PP-DC but not splenic DCs were able to convert retinol to retinoic acid. This work was the first to demonstrate that the migration of T cells to the gut is due to the retinoic acid secreted by gut-resident DCs, and also to confirm the importance of Vitamin A in gut immunology. It must be noted that the stromal cells in the MLN and PP also stained for RALDH, and so while it is clear that the DCs did produce retinoic acid, it is conceivable that stromal cells may contribute to this effect.
Studies have explored the function of gut DC loaded with commensal bacteria and the effect on IgA production (Macpherson, et al. (2001) supra). CD11c+ cells were purified from the PP and MLN of mice challenged with a known commensal (E. clocae). DC from mice gavaged with live bacteria demonstrated a rise in IgA+B cells and an increase in the production of IgA that was absent from mice treated with heat-killed bacteria. The increased response to live bacteria was not dependent on the presence of either B cells or T cells. DCs appear to be key in class switch recombination reaction to express IgA. Further studies in specific pathogen-free mice that were colonized with a simple flora demonstrated that there was a five-fold increase of IgA producing cells in the lamina propria following inoculation with high numbers of commensal bacteria of several different gram positive or negative strains. This enhanced IgA response to commensal inoculation was dependent on the uptake of the live bacteria that had penetrated the epithelial layer by CD11c+ DC and subsequent stimulation of B and T cell responses.
It has been suggested that commensal bacteria and the host immune system constitute an integrated defense system (Kitano & Oda (2006) Mol. Systems Biol. 2:2006.0022), wherein this symbiotic association has evolved to optimize its robustness against pathogen attacks and nutrient perturbations by harboring a broad range of microorganisms. Owing to the inherent propensity of a host immune system toward hyperactivity, it has been suggested that maintenance of bacterial flora homeostasis might be particularly important in the development of preventive strategies against immune disorders such as autoimmune diseases. While antibiotics such as minocycline (Zabad, et al. (2007) Mult. Scler. 13(4):517-26) and roxithromycin (Woessner, et al. (2006) Infection 34(6):342-4) have been evaluated, e.g., to determine systemic immunological changes and identify a causative connection between bacterial infections with C. pneumonia and multiple sclerosis, the effect of these antibiotics on commensal bacteria was not demonstrated.